The is a The biomedical discovery institute Microscopy dedicated to understanding the fundamental biology underlying Science health and disease. Its work Technology is helping to understand why disease develops and to translate Platform at discoveries into new ways to the Francis prevent, diagnose and treat illnesses such as cancer, heart Crick Institute disease, stroke, infections, and Lucy Collinson neurodegenerative diseases.

An independent organisation, its founding partners are the

Medical Research Council (MRC), Cancer Research UK,

Wellcome, University College London, Imperial College

London and King’s College London. The Crick was formed

in 2015, with many of the Crick’s scientists joining from two

‘parent’ institutes, the MRC’s National Institute for Medical

Research and Cancer Research UK’s London Research

Institute, and in 2016 it moved into a brand new state-of-

the-art building in central London which brings together

1500 scientists and support staff working collaboratively

across disciplines, making it the biggest biomedical research

facility under a single roof in Europe.

© Nick Guttridge

4 ISSUE 46 JUNE 2017 5 Each microscope room is a six-sided shielded box, • The Phenom-World DelPhi benchtop SEM with walls that contain complex metallic layers has an integrated fluorescence microscope to attenuate DC fields, and an active cancellation for correlative imaging system to attenuate AC fields. Under each • The FEI Twin 120 kV TEM has a cryo stage for microscope is a concrete platform, cast in place, and screening vitrified macromolecular samples supported by air springs that remove environmental prior to imaging on 200 kV and 300 kV TEMs vibration to <1 Hz. Each room has tight control of air • The FEI BioTwin 120 kV TEM has an quality, airflow, temperature stability and humidity, integrated iCorr fluorescence microscope for all of which are monitored through a complex correlative microscopy building management system with 27,000 individual • The FEI Quanta SEM has a Delmic SECOM monitoring points. In case of power outages, the integrated super-resolution fluorescence entire imaging suite is supported by a dedicated microscope for high accuracy correlative uninterruptible power supply, which was recently microscopy tested and proved invaluable during a power failure • The Zeiss Sigma and Merlin SEMs have Gatan at the local electricity sub-station. 3View stages for volume EM The time, effort, teamwork and expertise that went • The Zeiss Crossbeam 540 FIB SEM is used for into the project delivered an impressive suite of volume EM and also has a Leica cryo-stage for © Nick Guttridge rooms tailored to running sensitive high-resolution cryo-electron tomography sample preparation Building the Crick imaging experiments on a wide array of high-end • The Zeiss Versa 510 microCT has Atlas 5 • The total floor space is the size of 17.5 football fields instrumentation. software for 3D correlative imaging • There are over 1,500 rooms (twice as many as Buckingham Palace) Instrumentation Science and Technology • There are over 100 km of mains power cables installed (equivalent to the distance from The Structural Biology STP holds two FEI Titan Development London to Southampton) Krios transmission EMs (TEMs), for studying The EM STP collaborates with Crick researchers on • There are over 800 solar panels on the roof macromolecules and frozen hydrated cells. The EM ~80 projects at any one time. Some examples of our • The Crick was at one point the biggest single building construction project in the UK STP holds a range of microscopes that enable us to recent work are: study samples across scales, from single molecules • Studies of the role of RAD51 paralogs in facility. A sweet-spot for physical vibration, acoustic Facility design to whole model organisms. These include two repair of DNA damage, processes that are vibration and electro-magnetic fields was identified There are 14 Science Technology Platforms (STPs) benchtop scanning EMs (SEMs), two TEMs, three involved breast and ovarian cancer, with at the southwest corner of the site in the lower at the Crick, which concentrate expertise and SEMs, a Focused Ion Beam SEM and a microCT Simon Boulton’s lab using TEM (1, 2) basement, almost thirty metres underground. advanced equipment into teams that are accessible system. Each of these systems has additional • Studies of Mycobacterium tuberculosis infecting to all Crick researchers. Electron microscopes are A team of experts in electron microscopy, NMR, specialised functions: human lymphatic endothelial cells and housed in two of the STPs – Electron Microscopy advanced light microscopy, vibration control and (EM) and Structural Biology. The process of designing electromagnetic field control worked together with the Crick EM facilities began back in 2008, only a few the construction team, engineers and architects months after the land had been acquired and the over the next 8 years to deliver the project. This project officially announced. The challenge was to interaction was key to the success of the project, build a facility capable of holding high-end imaging and included frequent on-site visits to monitor the equipment, in a central London site surrounded complex build. by tube lines, roads, and the local, regional and The main construction phase finished in 2013 and international trains at St Pancras station. The a two-year period of fitting-out began. This phase site, an old railway storage yard to the west of St of the build included installation of measures to Pancras station and north of the British Library, was attenuate electromagnetic fields and vibrations. surveyed to identify the optimal position for the

6 ISSUE 46 JUNE 2017 7 macrophages with Max Gutierrez’s lab, using 3D correlative light and electron microscopy (CLEM) (3-5)

• Studies of B cell responses in the human immune system, with Facundo Batista’s lab, using SEM and volume EM (6-8)

The EM STP also develops new workflows. Some examples of our recent work are: • Development of sample preparation for Lucy's favourite image: Green fluorescent labelled lipids, localised to membranes in HeLa cells, imaged by integrated light and integrated light and electron microscopy (9- electron microscopy. 11) Favourite microscope: SEM, TEM, SBF SEM, • Development of a workflow for 3D CLEM FIB SEM, microCT – basically whichever one I’m using SBF SEM, used to study tuberculosis, HIV standing in front of at the time.... and cancer (5, 12) Favourite publication and why: The last • Development of cryo-correlative imaging one: ‘UltraLM and miniLM: Locator tools for smart of vitrified whole cells using synchrotron tracking of fluorescent cells in correlative light and radiation (13-16) electron microscopy’ {Brama, 2016 #832}, because it involves microscope hacking, and cuts across biology, microscopy, physics, optics, coding and Staff engineering. The EM STP team consists of nine postdoctoral If you weren’t a scientist, what would you scientists: seven are electron microscopists and be and why: If I could get paid for something I two have a background in physics, optics and image love but am not particularly good at, then a surfer! analysis. They are… What’s the best advice you’ve been Looking for an intelligent, automated Name: Lucy Collinson given: There’s no such thing as a stupid question Role: Head of EM STP and Microscopy Prototyping What three items would you take to a booking system for your Core Facility? desert island: Surfboard, insect repellant, luxury yacht Best-in-class online scheduling calendar with configurable rules to control who can book what, where and when.

A fully-featured reporting system for Would you like to see how Name: Raffaella Carzaniga generating billing and auditing information. Calpendo can benefit you and (known as Raffa) An integrated, intelligent system that allows your Facility? Contact us now Role: Deputy Head of the EM STP for multifaceted activity bookings. for a free demonstration and Automated and targeted emails for activity notifications, confirmations and cancellations. 30 day trial.

Qualifications: Degree and PhD in Microbiology, Brand new Inventory, Services and Workflow features for enhanced flexibility. post doc in Cell Biology Attending MMC 2017? Come and visit us at Stand 104 2006 Utilised in a multitude of Core Facilities across Joined the team: Europe, North America, Africa and Asia. Microscopy speciality: 3D correlative microscopy Tel: +44 (0)1235 813458 Email: [email protected]

8 ISSUE 46 JUNE 2017 Web: www.exprodo.com

infocus Exprodo advert 2017.indd 1 22/02/2017 16:16:26 Qualifications: MBiochem (Hons), PhD in What three items would you take to a If you weren’t a scientist, what would Telescope, comfortable armchair, neuroscience, post-docs in neuroscience and in desert island: you be and why: A vet because it combines good whisky peripheral nerve injury models science with my love of animals

Joined the team: 2011 What’s the best advice you’ve been given: The advice given to me during my PhD viva Microscopy speciality: CLEM, ILSEM, SBF SEM, Name: Anne Weston Senior Laboratory Research Scientist where one of the examiners suggested it would FIB SEM Role: be a good idea for me to get back into a career in Favourite microscope: Zeiss Crossbeam electron microscopy (!!!) 540 (new, shiny and extremely capable), and the What three items would you take to a venerable Jeol 1010 desert island: If we are talking purely material Raffa's favourite image: Negative staining of tobacco mosaic virus, imaged by transmission EM. Favourite publication and why: ‘Acute things then sun cream so that I don’t burn to a manipulation of diacylglycerol reveals roles in crisp, my kindle (fully charged of course) and a Qualification: PhD in Cell Biology nuclear envelope assembly & endoplasmic reticulum camera (with plenty of spare batteries) – this one Joined the team: 2012 morphology’ (17) – one of my first projects after is a bit of a cheat as it would be dual purpose in

Microscopy speciality: Jack of all trades joining the team, a steep learning curve, and an that I could use it to take photos but it would interesting group of people to work with also have all my favourite photos of my family still My first magnifying Favourite microscope: Qualifications: Degree in Zoology, PhD in stored in its memory! glass Bioinformatics I do not Favourite publication and why: Joined the team: 2003 have one – I like all of them Name: Matt Russell Multi-disciplinary but Microscopy speciality: Role: Senior Laboratory Research Scientist If you weren’t a scientist, what would probably more likely to be associated with SEM and Qualifications:MA/MSci in Biochemistry, PhD in you be and why: A Buddhist nun MicroCT Cell Biology What’s the best advice you’ve been Favourite microscope: SEM given: Better to be a small fish in a big pond than ‘Imaging a big fish in a small pond Favourite publication and why: transient blood vessel fusion events in zebrafish by What three items would you take to a correlative volume electron microscopy’ (18) – even desert island: A solar panel, a cable and my though I wasn’t involved in this publication I like it iPhone because it was really the start of the whole volume Christopher's favourite image: FIBing the moon’ – digging trenches in EM interest in our lab and that is a technique which resin for 3D imaging of cells using Focused Ion Beam SEM. Name: Christopher Peddie I particularly enjoy If you weren’t a scientist, what would you Role: Principle Laboratory Research Scientist be and why: Probably I’d focus on something more hands on like mechanical engineering since I seem to have a particular aptitude for that type of 2013 work and I find it interesting, but in another time Joined the team: and place (and with a bit more brilliance up top) I’d Microscopy speciality: 3D correlative light and have really liked to be a veterinary surgeon. Or a electron microscopy helicopter pilot. Favourite microscope: Philips EM400 What’s the best advice you’ve been Favourite publication and why: given: Controls are a prerequisite, question the ‘Mycobacterium tuberculosis replicates within dogma, and if you really want to find lla the typos, Anne's favourite image: 3D reconstruction of the fruit fly, image by necrotic human macrophages’ (3). Because it used read it backwards microCT.

10 ISSUE 46 JUNE 2017 11 Favourite microscope: Light microscope: Zeiss Qualifications: Undergraduate degree in LSM 710 inverted confocal microscope. Electron Physics with Electronics and Optoelectronics, microscope: FEI Tecnai G2 Spirit BioTWIN with MSc in Evolutionary and Adaptive Systems, Gatan Orius CCD camera DPhil in Experimental Atomic Physics (Quantum Information) Favourite publication and why: Without a doubt my postdoc paper, ‘Acute manipulation Joined the team: 2014, although I’ve been of diacylglycerol reveals roles in nuclear envelope interacting with the team since I saw Bram Koster assembly & endoplasmic reticulum morphology’ give a talk on CLEM in 2012 and started thinking (17) as this work not only marks the start of about building in situ CLEM systems. my collaboration with Lucy and Chris, but also Julia's favourite image: Microwave-processed cell, embedded for Image analysis and introduced me to CLEM, which led me to join the transmission EM in only two hours. Microscopy speciality: microscope hardware development team!! Microscopy speciality: High pressure freezing and electron tomography I don’t really use them If you weren’t a scientist, what would Favourite microscope: myself. The FIB SEM is definitely impressive. Also the Matt's favourite image: Revealing resin-embedded cells under a you be and why: Pastry chef! I love cakes and Favourite microscope: FEI Tecnai G2 Spirit platinum coat for correlative 3D EM using Serial Block Face SEM. smartphone microscope platforms that we use for the precision needed to make visually perfect cakes BioTWIN techniques for correlating LM, serial block face SEM, (picturing a French patisserie window...) outreach work (and are used by various Cancer and TEM that we’d predicted could be useful in an Favourite publication and why: ‘Membrane Research UK outreach teams around the country earlier methods paper remodeling during embryonic abscission in now) Caenorhabditis elegans’ (19). Because nothing is If you weren’t a scientist, what would better than combining live-cell imaging with high you be and why: Journalist, because I’ve always resolution ultrastructure wanted to try to understand the truth about things If you weren’t a scientist, what would What’s the best advice you’ve been A fashion designer, because I given: Do something that really interests you you be and why: like to make my own clothes What three items would you take to a My wife, my cast iron skillet, and Marie-Charlotte's favourite image: 3D correlative imaging of vitrified What’s the best advice you’ve been desert island: cells using cryo-soft X-ray tomography (with Liz Duke and colleagues a stereo microscope (Diamond Light Source) and Eva Perriero and colleagues (ALBA given: Relax! synchrotron)). What three items would you take to a What’s the best advice you’ve been desert island: Crisps, Crisps, Crisps Eat cake and carry on! Martin's favourite image: Automated reconstruction of the surface of a Name: Marie-Charlotte given: HeLa cell from Focused Ion Beam SEM data. Domart What three items would you take to Senior Favourite publication and why: ‘UltraLM Role: a desert island: a machete, a pot and a surf Name: Martin Jones Laboratory Research board! Role: Deputy Head of Microscopy Prototyping and miniLM: Locator tools for smart tracking of Scientist fluorescent cells in correlative light and electron Qualifications: microscopy’ (11), which is the realisation of an idea I Degree in Cell Biology Name: Julia had and randomly pitched to Lucy, the development and Physiology, Konig of the project is why I ended up in the EM STP Masters in Oncology, Senior Role: If you weren’t a scientist, what would you PhD in Biochemistry Laboratory Research be and why: Teacher. Before my degree I was and Molecular Biology, Scientist more or less planning to go into teaching, I think Postdoc in Cell Qualifications: PhD it’s one of the most important but unfortunately Biology in Cell Biology underappreciated jobs. During my DPhil and physics Joined the team: September 2013 Joined the team: postdocs I taught various courses and labs and really Microscopy speciality: Correlative light and 2016 enjoyed the challenge of having to explain things in soft X-ray or electron microscopy

12 ISSUE 46 JUNE 2017 13 weeks of launch, Etch a Cell had more than 1000 different ways for different students, it really makes and miniLM: Locator tools for smart tracking of 2) Automated image analysis Development of automated image analysis volunteers and more than 13,000 nuclear envelope sure you understand it yourself! fluorescent cells in correlative light and electron segmentations. The first average models of nuclear microscopy’ (11), because it was my first paper algorithms for EM is challenging. Many cell envelopes are now being made and analysed, and What’s the best advice you’ve been where I felt like I knew what I was talking about membranes have similar grey values in EM images, given: My old history teacher at school was If you weren’t a scientist, what would and so simple histogram thresholding often gives a worried that I was too shy and couldn’t hold you be and why: A carpenter, or a baker, or an poor result when trying to select a subset of cell conversations very well, so he told me: whenever architect, or a pro triathlete. Because I like making organelles. Manual annotation (segmentation) of someone asks you a question, think of what their things, buildings, and riding my bike organelles is usually required to create accurate follow up question might be and try to answer that What’s the best advice you’ve been 3D models, but the process is extremely time- too, before they ask. I think this approach works given: If it’s not ok, it’s not the end consuming. Machine learning techniques are starting very well in science too, following a line of reasoning to deliver semi-automated image analysis algorithms, along to its logical conclusion is important What three items would you take to a particularly in the field of connectomics. However, A boat to get away, food and desert island: machine learning requires large amounts of ‘ground What three items would you take to a water for the journey home. desert island: MP3 player with all my music, truth’ training data, which is lacking in EM because Crocodile Dundee knife and some sort of tent/ of the time taken for expert manual segmentation. shelter Future Directions will be used to train computers via deep learning Now that EM image acquisition is becoming more algorithms. Once you have finished reading infocus, automated, one of the biggest challenges in our Name: Lizzy Brama we hope you will grab a cup of tea, and stop by the work is handling and analysing the huge amounts of Etch a Cell website to add a few segmentations of Role: Senior Laboratory Research Scientist data we produce. Volume EM techniques, including your own… Degree in Physics, PhD in Qualifications: SBF SEM and FIB SEM, can turn out thousands of experimental quantum optics images in just a few days. Two approaches we are Further Information pursuing to deal with the data deluge are: If you would like to visit us and hear more about the impact of EM on biomedical research, 1) Smart data acquisition you can register for our Crick EM Opening Correlative microscopy may be seen as a form Symposium. The symposium will take place on th th of smart data acquisition, in that cells of interest July 12 and 13 2017, and will feature talks are identified using one imaging modality (e.g. from international experts on EM imaging across fluorescence microscopy) and then high resolution scales, from molecules to whole organisms. The images are collected using a second imaging modality symposium will be accompanied by a day of workshops on July 14th. Registration can be found (e.g. electron microscopy). Introducing miniaturised Fiona Hanson/Francis Crick Institute at www.rms.org.uk/crick-symposium-2017. fluorescence microscopes into volume EMs will allow us to track fluorescent cells on-the-fly during Website: www.crick.ac.uk/research/science- automated data acquisition runs, so that we only To deal with this problem, we are harnessing the technology-platforms/electron-microscopy collect images from the cells and tissues of interest. brute force power of Citizen Science to produce 2014 Twitter: @EM_STP and @EtchACell Joined the team: Miniaturisation is required due to the extremely ground truth segmentations. Our project ‘Etch a Building them Email: [email protected] Microscopy speciality: tight space within the SEM chamber in these Cell’ (www.zooniverse.org/projects/h-spiers/etch- Till Photonics iMic, Favourite microscope: systems. Our prototype fluorescence microscope, a-cell) is hosted by the Zooniverse platform, which You can view more information about the more people ought to have bought them the miniLM, is only 2.8 mm in diameter (11). has more than a million volunteer citizen scientists Francis Crick Institute on the RMS Facilities Favourite publication and why: ‘UltraLM working on projects from the arts to ecology, Database at www.rms.org.uk/facilities-database. and from space to medicine. Etch a Cell asks Further information about Etch a Cell is citizen scientists to draw around cell organelles, available at www.zooniverse.org/projects/h- starting with the nuclear envelope. Within two spiers/etch-a-cell. Lizzy's favourite image: Cavity-induced power broadening ions.

14 ISSUE 46 JUNE 2017 15 References: cell responses. Science (New York, N.Y.) 355, 641-647 (2017). 1. M. R. Taylor et al., A Polar and Nucleotide-De- labtech... pendent Mechanism of Action for RAD51 9. C. J. Peddie et al., Correlative and integrated Paralogs in RAD51 Filament Remodeling. Mo- light and electron microscopy of in-resin GFP lecular cell , 926-939 (2016). 64 fluorescence, used to localise diacylglycerol in ...innovative and practical tools mammalian cells. Ultramicroscopy, (2014). 2. M. R. Taylor et al., Rad51 Paralogs Remodel Pre-synaptic Rad51 Filaments to Stimulate Ho- for electron microscopists 10. C. J. Peddie, N. Liv, J. P. Hoogenboom, L. M. Col- mologous Recombination. Cell , 271-286 162 linson, Integrated Light and Scanning Electron (2015). Microscopy of GFP-Expressing Cells. Methods in cell biology , 363-389 (2014). 3. T. R. Lerner et al., Mycobacterium tuberculosis 124 replicates within necrotic human macrophages. 11. E. Brama et al., ultraLM and miniLM: Locator J Cell Biol , 583-594 (2017). 216 tools for smart tracking of fluorescent cells in correlative light and electron microscopy. Well- 4. T. R. Lerner et al., Lymphatic endothelial cells come Open Res , 26 (2016). are a replicative niche for Mycobacterium 1 tuberculosis. The Journal of clinical investigation 12. D. O. Nkwe, A. Pelchen-Matthews, J. J. Burden, , 1093-1108 (2016). 126 L. M. Collinson, M. Marsh, The intracellular plasma membrane-connected compartment in 5. M. R. Russell et al., 3D correlative light and Micro to Nano the assembly of HIV-1 in human macrophages. electron microscopy of cultured cells using consumables BMC biology , 50 (2016). serial blockface scanning electron microscopy. 14 Journal of cell science , 278-291 (2017). 130 13. R. Carzaniga, M. C. Domart, E. Duke, L. M. Collinson, Correlative cryo-fluorescence and 6. M. Burbage et al., Cdc42 is a key regulator of B cryo-soft x-ray tomography of adherent cells cell differentiation and is required for antiviral at European synchrotrons. Methods in cell biolo- Pie Scientific humoral immunity. Journal of Experimental Med- gy 124, 151-178 (2014). plasma cleaners icine In Press, (2014). 14. R. Carzaniga, M. C. Domart, L. M. Collinson, E. 7. O. Thaunat et al., Asymmetric segregation of Duke, Cryo-soft X-ray tomography: a journey polarized antigen on B cell division shapes into the world of the native-state cell. Proto- presentation capacity. Science (New York, N.Y.) plasma, (2013). 335, 475-479 (2012). Kleindiek manipulators DiATOME 15. E. Duke, K. Dent, M. Razi, L. M. Collinson, Bio- and nano tools diamond knives 8. N. Martinez-Martin et al., A switch from ca- logical applications of cryo-soft X-ray tomog- nonical to noncanonical autophagy shapes B raphy. Journal of microscopy 255, 65-70 (2014).

16. E. M. H. Duke et al., Imaging endosomes and autophagosomes in whole mammalian cells Safematic using correlative cryo-fluorescence and cryo- vacuum coaters soft X-ray microscopy (cryo-CLXM). Ultrami- croscopy, (2013). 17. M. C. Domart et al., Acute manipulation of • Consumables and accessories for SEM, FIB and TEM diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morpholo- gy. PloS one 7, e51150 (2012). • EM specimen preparation and manipulation 18. H. E. Armer et al., Imaging transient blood • Established UK-wide sales and service vessel fusion events in zebrafish by correlative volume electron microscopy. PloS one 4, e7716 Friendly, knowledge-based support (2009). •

19. J. Konig, E. B. Frankel, A. Audhya, T. Muller-Re- • Serving UK science for 24 years ichert, Membrane remodeling during embry- onic abscission in Caenorhabditis elegans. J Cell Biol, (2017). Fiona Hanson/Francis Crick Institute labtech ELECTRON MICROSCOPY DIVISION www.labtech-em.com Tel: 01825 744690 16 ISSUE 46 JUNE 2017